Expression constructs and methods of genetically engineering methylotrophic yeast

Patent metadata

Field	Value
Publication number	US-9938327-B2
Application number	US-201715678891-A
Country	US
Kind code	B2
Filing date	Aug 16, 2017
Priority date	May 11, 2015
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

Methods and materials for genetically engineering methylotrophic yeast are provided.

First claim

What is claimed is:

1. A methylotrophic Pichia yeast cell comprising:
a nucleic acid molecule having at least 90% sequence identity to SEQ ID NO:5 and encoding a leghemoglobin polypeptide operably linked to a promoter element from P. pastoris and a Mxr1 transcriptional activator sequence from P. pastoris ; and
a nucleic acid molecule having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:11, 12, 13, 14, 15, 16, 17, and 18 and encoding at least one polypeptide involved in heme biosynthesis operably linked to a promoter element from P. pastoris and a Mxr1 transcriptional activator sequence from P. pastoris.

2. The yeast cell of claim 1 , wherein the nucleic acid molecule encoding the leghemoglobin polypeptide has at least 95% sequence identity to the sequence shown in SEQ ID NO:5.

3. The yeast cell of claim 1 , wherein the nucleic acid molecule encoding the leghemoglobin polypeptide has the sequence shown in SEQ ID NO:5.

4. The yeast cell of claim 1 , wherein the leghemoglobin polypeptide has the sequence shown in SEQ ID NO:6.

5. The yeast cell of claim 1 , wherein the nucleic acid molecule encoding at least one polypeptide involved in heme biosynthesis has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, and 18.

6. The yeast cell of claim 1 , wherein the nucleic acid molecule encoding at least one polypeptide involved in heme biosynthesis has a sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, and 18.

7. The yeast cell of claim 1 , wherein the promoter element from P. pastoris is a constitutive promoter element from P. pastoris or a methanol-inducible promoter element from P. pastoris.

8. The yeast cell of claim 7 , wherein the constitutive promoter element from P. pastoris is a promoter element from a transcriptional elongation factor EF-1 (TEF1) gene.

9. The yeast cell of claim 8 , wherein the TEF promoter element has the nucleic acid sequence shown in SEQ ID NO:10.

10. The yeast cell of claim 7 , wherein the methanol-inducible promoter element from P. pastoris is an alcohol oxidase I (AOX1) promoter element.

11. The yeast cell of claim 10 , wherein the AOX1 promoter element has the nucleic acid sequence shown in SEQ ID NO:7.

12. The yeast cell of claim 1 , wherein the promoter element from P. pastoris comprises a constitutive promoter element from P. pastoris and a methanol-inducible promoter element from P. pastoris.

13. The yeast cell of claim 12 , wherein the constitutive promoter element from P. pastoris is a promoter element from a transcriptional elongation factor EF-1 (TEF1) gene.

14. The yeast cell of claim 13 , wherein the TEF promoter element has the nucleic acid sequence shown in SEQ ID NO:10.

15. The yeast cell of claim 12 , wherein the methanol-inducible promoter element from P. pastoris is an alcohol oxidase I (AOX1) promoter element.

16. The yeast cell of claim 15 , wherein the AOX1 promoter element has the nucleic acid sequence shown in SEQ ID NO:7.

17. The yeast cell of claim 1 , wherein the Mxr1 transcriptional activator sequence from P. pastoris has the nucleic acid sequence shown in SEQ ID NO:1.

18. The yeast cell of claim 1 , wherein the Mxr1 transcriptional activator sequence from P. pastoris has the amino acid sequence shown in SEQ ID NO:2.

19. The yeast cell of claim 1 , wherein the nucleic acid molecule encoding the leghemoglobin is present in the yeast cell in at least two copies.

20. The yeast cell of claim 1 , wherein the nucleic acid molecule encoding the leghemoglobin operably linked to the promoter element from P. pastoris and the Mxr1 transcriptional activator sequence from P. pastoris further comprises a termination sequence.

21. The yeast cell of claim 1 , wherein the nucleic acid encoding the at least one polypeptide involved in heme biosynthesis operably linked to the promoter element from P. pastoris and the Mxr1 transcriptional activator sequence from P. pastoris further comprises a termination sequence.

Assignees

• Impossible Foods Inc

Inventors

• Shankar Smita
• Hoyt Martin Andrew

Classifications

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12N15/81

  for yeasts · CPC title

• C12N15/815 Primary

  for yeasts other than Saccharomyces · CPC title

• C07K14/39 Primary

  from yeasts · CPC title

• C07K14/805

  Haemoglobins; Myoglobins · CPC title